Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans-a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.

The ins and outs of vanillyl alcohol oxidase: Identification of ligand migration paths.

Vanillyl alcohol oxidase (VAO) is a homo-octameric flavoenzyme belonging to the VAO/PCMH family. Each VAO subunit consists of two domains, the FAD-binding and the cap domain. VAO catalyses, among other reactions, the two-step conversion of p-creosol (2-methoxy-4-methylphenol) to vanillin (4-hydroxy-3-methoxybenzaldehyde). To elucidate how different ligands enter and exit the secluded active site, Monte Carlo based simulations have been performed. One entry/exit path via the subunit interface and two additional exit paths have been identified for phenolic ligands, all leading to the si side of FAD. We argue that the entry/exit path is the most probable route for these ligands. A fourth path leading to the re side of FAD has been found for the co-ligands dioxygen and hydrogen peroxide. Based on binding energies and on the behaviour of ligands in these four paths, we propose a sequence of events for ligand and co-ligand migration during catalysis. We have also identified two residues, His466 and Tyr503, which could act as concierges of the active site for phenolic ligands, as well as two other residues, Tyr51 and Tyr408, which could act as a gateway to the re side of FAD for dioxygen. Most of the residues in the four paths are also present in VAO's closest relatives, eugenol oxidase and p-cresol methylhydroxylase. Key path residues show movements in our simulations that correspond well to conformations observed in crystal structures of these enzymes. Preservation of other path residues can be linked to the electron acceptor specificity and oligomerisation state of the three enzymes. This study is the first comprehensive overview of ligand and co-ligand migration in a member of the VAO/PCMH family, and provides a proof of concept for the use of an unbiased method to sample this process.

Assessment of deoxyhypusine hydroxylase as a putative, novel drug target.

Antimalarial drug resistance has nowadays reached each drug class on the market for longer than 10 years. The focus on validated, classical targets has severe drawbacks. If resistance is arising or already present in the field, a target-based High-Throughput-Screening (HTS) with the respective target involves the risk of identifying compounds to which field populations are also resistant. Thus, it appears that a rewarding albeit demanding challenge for target-based drug discovery is to identify novel drug targets. In the search for new targets for antimalarials, we have investigated the biosynthesis of hypusine, present in eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine hydroxylase (DOHH), which has recently been cloned and expressed from P. falciparum, completes the modification of eIF5A through hydroxylation. Here, we assess the present druggable data on Plasmodium DOHH and its human counterpart. Plasmodium DOHH arose from a cyanobacterial phycobilin lyase by loss of function. It has a low FASTA score of 27 to its human counterpart. The HEAT-like repeats present in the parasite DOHH differ in number and amino acid identity from its human ortholog and might be of considerable interest for inhibitor design.

Preliminary assessment of the C13-side chain 2'-hydroxylase involved in taxol biosynthesis.

The biosynthesis of the anticancer drug Taxol in yew (Taxus) species is thought to involve the preliminary formation of the advanced taxane diterpenoid intermediate baccatin III upon which the functionally important N-benzoyl phenylisoserinoyl side chain is subsequently assembled at the C13-O-position. In vivo feeding studies with Taxus tissues and characterization of the two transferases responsible for C13-side chain construction have suggested a sequential process in which an aminomutase converts alpha-phenylalanine to beta-phenylalanine which is then activated to the corresponding CoA ester and transferred to baccatin III to yield beta-phenylalanoyl baccatin III (i.e., N-debenzoyl-2'-deoxytaxol) that undergoes subsequent 2'-hydroxylation and N-benzoylation to afford Taxol. However, because the side chain transferase can utilize both beta-phenylalanoyl CoA and phenylisoserinoyl CoA in the C13-O-esterification of baccatin III, ambiguity remained as to whether the 2'-hydroxylation step occurs before or after transfer of the amino phenylpropanoyl moiety. Using cell-free enzyme systems from Taxus suspension cells, no evidence was found for the direct hydroxylation of beta-phenylalanine to phenylisoserine; however, microsomal preparations from this tissue appeared capable of the cytochrome P450-mediated hydroxylation of beta-phenylalanoyl baccatin III to phenylisoserinoyl baccatin III (i.e., N-debenzoyltaxol) as the penultimate step in the formation of Taxol and related N-substituted taxoids. These preliminary results, which are consistent with the proposed side chain assembly process, have clarified an important step of Taxol biosynthesis and set the foundation for cloning the responsible cytochrome P450 hydroxylase gene.

The TIGRFAMs database of protein families.

TIGRFAMs is a collection of manually curated protein families consisting of hidden Markov models (HMMs), multiple sequence alignments, commentary, Gene Ontology (GO) assignments, literature references and pointers to related TIGRFAMs, Pfam and InterPro models. These models are designed to support both automated and manually curated annotation of genomes. TIGRFAMs contains models of full-length proteins and shorter regions at the levels of superfamilies, subfamilies and equivalogs, where equivalogs are sets of homologous proteins conserved with respect to function since their last common ancestor. The scope of each model is set by raising or lowering cutoff scores and choosing members of the seed alignment to group proteins sharing specific function (equivalog) or more general properties. The overall goal is to provide information with maximum utility for the annotation process. TIGRFAMs is thus complementary to Pfam, whose models typically achieve broad coverage across distant homologs but end at the boundaries of conserved structural domains. The database currently contains over 1600 protein families. TIGRFAMs is available for searching or downloading at www.tigr.org/TIGRFAMs.

Identification of lactate. Analytical methods of pharmacopoeias with DBH in respect to environmental and economical concern, Part 4.

The identification of lactate according to Ph. Eur. 1997 and DAB 2000 uses the oxidation of lactic acid to pyruvic acid by boiling with bromine water in sulphuric acid. Acetaldehyde arising by decarboxylation is detected according to Legal applying a time consuming and troublesome procedure. 1,3-Dibromo-5,5-dimethylhydantoin (DBH) as well as potassium bromate can replace elemental bromine. Lactic acid and all lactates of Ph. Eur. 1997 and DAB 2000 can be identified better and faster using lactate oxidase (LOD, test strip system Accusport). According to DAB 2000 the base of ethacridine lactate has to be separated. This is no longer necessary, if an enzymatic identification is applied.

Homology modeling by the ICM method.

Five models have been built by the ICM method for the Comparative Modeling section of the Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. The targets have homologous proteins with known three-dimensional structure with sequence identity ranging from 25 to 77%. After alignment of the target sequence with the related three-dimensional structure, the modeling procedure consists of two subproblems: side-chain prediction and loop prediction. The ICM method approaches these problems with the following steps: (1) a starting model is created based on the homologous structure with the conserved portion fixed and the nonconserved portion having standard covalent geometry and free torsion angles; (2) the Biased Probability Monte Carlo (BPMC) procedure is applied to search the subspaces of either all the nonconservative side-chain torsion angles or torsion angles in a loop backbone and surrounding side chains. A special algorithm was designed to generate low-energy loop deformations. The BPMC procedure globally optimizes the energy function consisting of ECEPP/3 and solvation energy terms. Comparison of the predictions with the NMR or crystallographic solutions reveals a high proportion of correctly predicted side chains. The loops were not correctly predicted because imprinted distortions of the backbone increased the energy of the near-native conformation and thus made the solution unrecognizable. Interestingly, the energy terms were found to be reliable and the sampling of conformational space sufficient. The implications of this finding for the strategies of future comparative modeling are discussed.